DB - L5 - Cell-cell interactions - TGFB and FGF signalling

what is the main way that cells communicate with each other?

through paracrine signalling

what is paracrine signalling?

• when a cell secretes signalling molecules that act on nearby cells

• the ligand (signal) produced by a cell navigates through the extracellular space and acts on nearby cells

• it binds on the cell membrane - this binding triggers a variety of molecular events which leads to cellular responses

• these responses occur through a signal transduction

what is a signal transduction?

• he process by which a cell takes an external signal (like a ligand binding to a receptor) and converts it into an internal response

what are the 3 common features in a signal transduction pathway?

1. reception

2. transduction

3. response

explain a simple signal transduction pathway

1. reception - when a ligands binds to a cell surface receptor and activates it

2. transduction - the activation of the receptor activates a cascade of secondary messengers - which carry signal from membrane to nucleus

3. response - a transcriptional factor (TF) is activated and induces the transcription of specific target genes (cell changes its behaviour)

what is the TGF-B superfamily?

• consists of over 30 dimeric ligands

• splits into 2 main branches

  → TGF-B-like family

  → BMP-like family

what are the two branches that the TGF-B superfamily is divided into?

1. TGF-B-like family

2. BMP-like family

what are the key roles of the TGF-B-like subfamily?

• regulate cell fate, growth, and patterning

• establish morphogen gradients

• control proliferation, differentiation, apoptosis

• specify positional identity of cells

• influence ECM production - by telling cells to make more or less of certain ECM components

what are the key roles of the BMP-like subfamily?

• bone-morphogenic proteins

• establish dorsal-ventral patterning

• control cell fate decisions

• regulate bone, cartilage, and organ formation

• influence cell proliferation, differentiation, and apoptosis

what Smad do the TGF-B signal through?

• Smad 2/3

what Smad do the BMP signal through?

• Smad 1/5/8

what is the similarity between TGF-B family and BMP family?

• they both utilise Smad 4 (coSmad)

• they have distinct receptors and Smads - but they work similarly

what are TGF-B receptors?

serine/threonine kinases

what does the ligand-receptor complex of Smad signalling consist of?

• dimeric ligand

• 2x Type 1 receptors

• 2x Type 2 receptors

explain the reception stage of Smad signalling

1. TGF-B/BMP ligands come along and bind to Type 2 receptors

2. once bound to Type 2 receptors - they recruit Type 1 receptors to the complex

3. once assembled the Type 2 receptors activate the Type 1 receptors by phosphorylation of multiple serine/threonine in the GS domain

explain the transduction stage of Smad signalling

• once the receptors are activated - Type 1 receptors activate the Smad proteins through phosphorylation

• the Smad proteins act as secondary messengers

explain the response stage of Smad signalling?

• tissue-specific TFs modulate Smad binding to target genes

what is a common theme in signal transduction?

• components of the pathway change their subcellular localisation to activate the pathway

how is control of BMP signalling operated?

• through inhibition - a large class of inhibitors

• to ensure BMP signal is activated only at the right time and place

• some of these inhibitors act as ligand traps

  → they prevent ligands from binding and activating the receptors

• examples of BMP inhibitors = chordin, noggin, follistatin

how can we visualise BMP pathway activity?

• by using an antibody against the phosphorylated Smad proteins

• the (red) signal in the immunofluorescence corresponds to the phosphorylated Smad 1/5 (pSmad 1/5) - active BMP signalling

• this means its a readout of the BMP Smad pathway

immunofluorescence in zebrafish tailbud - BMP detection

• red patch = have active BMP signalling

• green patch = chordin and noggin (inhibitors)

• there’s no red signal where there’s chordin and noggin - because they are inhibitors so they prevent those cells from responding to BMPs

what are the tools for analysis of TGF-B signalling?

• genetic engineering to generate DNA that encodes a mutated receptor (functional modifications)

• the DNA is then expressed in cells/embryo and interferes with signalling

what are 2 examples of functional modifications for analysis of TGF-B signalling?

1. Kinase dead - introduce an amino acid change which stops kinase domain from working = loss of function

   • when receptor expressed in embryo = blocks BMP signalling

2. Constitutively active - generate a constitutively active receptor - always active - done by mutating an amino acid = gain of function

   • forces Smad activation without ligand binding

what does the existence of several inhibitors allow?

• allows for fine tuning of the signal output

• this is an important feature of cell fate specification in early embryogenesis

what are Receptor Tyrosine Kinases (RTKs)?

• large family of cell surface signalling receptors

• role in cell migration, proliferation, differentiation, survival

• 58 RTK genes grouped into 20 subfamilies

• some receptors are ligand specific while others interact with many ligands

• exist as monomers when unbound

what are the domains of RTKs?

• extracellular domains - determine specificity of ligand binding

• transmembrane domain

• intracellular domains - contain tyrosine kinase domains

  → they have kinase activity and so can phosphorylate proteins

explain RTK receptor activation

1. receptor is activated when ligand binds to the receptor

2. the ligands come together and dimerise - facilitating receptor dimerisation

3. the dimerisation brings together the intracellular tyrosine kinase domains of the subunits

4. once positioned - the kinase domains cross-phosphorylate each other

5. as result this:

   • increases the activity of kinases

   • stabilises the receptor in the active state

   • causes the kinase to phosphorylate other tyrosines in the receptor - to create docking sites

what are docking sites?

• phosphorylation sites on activated receptors

• these sites are sticky to various other proteins

• other proteins come along and bind to these phosphorylated sites

• phosphorylated tyrosines act as docking sites for downstream signalling proteins

what is Ras?

• a small GTPase that acts as an intracellular switch

• it exists in 2 states - inactive or active

• if Ras is bound to GDP = inactive state

• if Ras is bound to GTP = active state

• GAPs and GEFs govern whether Ras is GDP (inactive) or GTP (active) bound

what are GEFs?

• guanine nucleotide exchange factors

• they replace GDP with GTP

what are GAPs?

• GTPase activating proteins

• they hydrolyse GTP into GDP - removing the Pi (phosphate) group

what is Raf?

• a serine/threonine protein kinase that works downstream of Ras

• downstream effector protein

• when Ras interacts with Raf - this activates Raf’s serine threonine kinase activity

explain signal transduction - the Ras pathway

1. ligand binds RTK - receptor dimerises and autophosphorylates tyrosines

2. GRB2 have 2 domains

   • SH2 domain binds phosphorylated tyrosines

   • SH3 domain binds SOS (GEF protein)

3. SOS switches Ras from Ras-GDP to Ras-GTP

4. Ras-GTP activates Raf

5. Raf activates MEK

6. MEK activates MAPK (ERK)

7. MAPK (ERK) goes into nucleus - causing changes in gene expression - drives cell growth, division, survival

what are the 2 ways activated MAPK can affect cell behaviour?

1. directly phosphorylates TFs

   → altering transcription causing changes in genes expressed in cell

2. phosphorylates other secondary messengers

what is the family of FGF ligands?

• family of ligands that bind to one subfamily of RTKs - the FGF receptors

• FGFR1, FGFR2, FGFR3, FGFR4 - all FGFs signal through these receptors

• 22 members

what are the 3 subfamilies the 22 members of the FGF ligands split into?

1. paracrine FGF

2. intracrine FGF

3. endocrine FGF

what are paracrine FGFs?

• monomeric ligands secreted by the cell and bind to receptors on neighbouring cells

what are the roles of FGF8?

• limb formation

• mesoderm formation

• midbrain/hindbrain boundary

explain the structure of the FGF receptor?

• extracellular domain - made up of 3 immunoglobulin domains: D1, D2, D3

• acid box domain - in between D1 and D2

  → represses receptor activity in absence of ligand

• heparin binding site in D2 domain

• D2 and D3 = responsible for ligand binding

• small transmembrane domain

• intracellular kinase domain

what does FGFR activation by paracrine FGFs require?

• the formation of a large complex with molecules called heparin sulphate proteoglycans (HSPGs)

what are HSPGs (heparan sulphate proteoglycans)?

• extracellular modifiers of cell-cell signalling

• protein core: transmembrane, tethered, or secreted

• each sugar (heparan) can be modified in many ways - especially sulphation

• modification can result in a code - that creates binding sites for specific proteins

• sulphate domains are polyanionic (negative charge)

paracrine FGFs and HSPGs

• paracrine FGFs have high affinity for HSPGs

• they are retained and act locally

endocrine FGFs and HSPGs

• endocrine FGFs have low affinity for HSPGs

• they diffuse into blood stream

how can FGF signalling trigger different cell behaviours?

through distinct pathways

• Mapk (TF - FOS) - cell proliferation

• Akt (TF - FOXO) - cell survival

• Calcineurin (TF - NFAT) - cell motility

what are human diseases associated with mutations in FGFR?

• Apert syndrome

• Achondroplasia

• Pfeiffer syndrome